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The contribution of microglia to neuronal circuit refinement and pruning during development has been the subject of intensive investigation in the past few years. Numerous lines of evidence imply that microglia may actively phagocytose bits of developing neuronal processes, including synapses, through a behavior that has been described as “nibbling” or trogocytosis. Influential electron micrographic studies have found probable synaptic material localized within microglial phagocytic compartments. However, as microglia are well known to ravenously remove debris left behind by apoptotic cells in the developing CNS, such evidence from snapshots of fixed microglia is far from conclusive. Other studies have shown circuit development defects in transgenic animals in which microglial phagocytic activity has been suppressed. But given the many signaling molecules known to be released by microglia, it is difficult to know with certainty whether the defect reflects the participation of myeloid cells in synaptic trogocytosis or in more conventional local interactions involving the release of chemical signals. Finally, ex vivo slice culture preparations have offered more direct evidence for synaptic nibbling, but the highly invasive process of preparing such brain slices may not accurately reflect the physiological activity of microglia in a healthy developing brain. The retinotectal system of the <i>Xenopus laevis</i> tadpole is a classic model for studying the activity-dependent refinement of the developing visual system due to the ease with which <i>in vivo</i> imaging and visually-evoked behavioral studies can be performed in intact translucent tadpoles without the need for any inflammation-inducing surgical procedures. In the current study, we have exploited this simple <i>in vivo </i>model to directly follow microglia and neurons over many hours as they interact within the developing retinotectal circuit. Our results include direct observation and quantification of the uptake of synaptic material from healthy, remodeling axons by microglia over time. We further went on to test the consequences of microglial depletion in circuit development and found evidence for both structural exuberance as well as the disruption of visually-mediated behaviors. Finally, we examined the hypothesis that complement C3 may in part underlie the microglia-mediated structural remodeling of retinotectal axons by showing that the endogenously expressed complement inhibitor protein CD46 appears to promote arbor elaboration whereas decorating axons with C3 causes a failure to ramify arbors within the optic tectum.This <i>in vivo</i> study is the first, to our knowledge, to directly observe and quantify the trogocytosis of presynaptic material by microglia in the intact developing central nervous system. We introduce a novel and highly efficient assay for testing visuomotor behaviors in Xenopus tadpoles. And our study identifies complement C3 and its inhibition by the complement regulator protein CD46 as important regulators of axonal pruning by microglia in circuit development. We believe that this work constitutes a major advance both by conclusively demonstrating and by mechanistically investigating how microglia participate in circuit refinement during development.

近年来，小胶质细胞（microglia）在发育过程中对神经元环路重塑与突触修剪的调控作用，已成为学界备受关注的深度研究课题。大量研究证据表明，小胶质细胞可通过一种被称为“啃噬”或胞啃作用（trogocytosis）的行为，主动吞噬发育中的神经元突起片段，包括突触结构。具有影响力的电子显微成像研究发现，小胶质细胞的吞噬囊泡内存在疑似突触物质的定位分布。然而，由于学界早已熟知小胶质细胞会高效清除发育中枢神经系统（central nervous system, CNS）中凋亡细胞遗留的碎片，因此仅基于固定状态下小胶质细胞的静态成像证据，远不足以得出确定性结论。另有研究显示，在小胶质细胞吞噬活性被抑制的转基因动物中，出现了环路发育缺陷。但鉴于小胶质细胞可分泌多种信号分子，目前难以确切判断该缺陷究竟是髓系细胞（myeloid cells）参与突触胞啃作用所致，还是源于更常见的局部化学信号释放介导的相互作用。最后，离体脑片培养（ex vivo slice culture）实验为突触啃噬现象提供了更为直接的证据，但制备此类脑片的高侵入性操作，可能无法准确反映健康发育大脑中小胶质细胞的生理活性。非洲爪蟾（Xenopus laevis）蝌蚪的视网膜顶盖系统（retinotectal system）是研究发育中视觉系统活动依赖性重塑的经典模型，这是因为完整透明的蝌蚪可直接进行活体（in vivo）成像与视觉诱发行为学研究，且无需引发炎症的外科操作。在本研究中，我们利用这一简便的活体模型，对发育中的视网膜顶盖环路内相互作用的小胶质细胞与神经元进行了长达数小时的连续追踪。研究结果包括随时间推移，小胶质细胞摄取健康、正在重塑的轴突（axon）突触物质的直接观察与定量分析。我们进一步检测了小胶质细胞耗竭对环路发育的影响，结果发现既存在结构过度增生的现象，也伴随视觉介导行为的紊乱。最后，我们针对补体C3（complement C3）可能部分介导小胶质细胞对视网膜顶盖轴突的结构重塑这一假说展开了验证：实验表明，内源性表达的补体抑制蛋白（complement inhibitor protein）CD46可促进轴突分支生长，而在轴突表面修饰补体C3则会导致其在视顶盖（optic tectum）内无法形成分支。据我们所知，这项活体研究首次在完整的发育中枢神经系统中，直接观察并定量分析了小胶质细胞对突触前物质的胞啃作用。我们还建立了一种新颖且高效的非洲爪蟾蝌蚪视觉运动行为检测方法。本研究证实补体C3及其受补体调节蛋白CD46的调控，是小胶质细胞在发育环路中介导轴突修剪的关键调控因子。我们认为，本研究不仅明确证实了小胶质细胞如何参与发育过程中的环路重塑，还从机制层面展开了深入探究，因此具有重要的科学推进意义。